The commercial inulinase obtained from Aspergillus niger was non-covalently immobilized on multiwalled carbon nanotubes (MWNT-COOH). The immobilization conditions for the carbon nanotubes were defined by the central composite rotational design (CCRD). The effects of enzyme concentration (0.8%–1.7% v/v) and adsorbent:adsorbate ratio (1:460–1:175) on the enzyme immobilization were studied. The adsorbent:adsorbate ratio variable has positive effect and the enzyme concentration has a negative effect on the inulinase immobilization (U/g) response at the 90% significance level. These results show that the lower the enzyme concentration and the higher the adsorbent:adsorbate ratio, better is the immobilization. According to the results, it is possible to observe that the carbon nanotubes present an effective inulinase adsorption. Fast adsorption in about six minutes and a loading capacity of 51,047 U/g support using a 1.3% (v/v) inulinase concentration and a 1:460 adsorbent:adsorbate ratio was observed. The effects of temperature on the immobilized enzyme activity were evaluated, showing better activity at 50 °C. The immobilized enzyme maintained 100% of its activity during five weeks at room temperature. The immobilization strategy with MWNT-COOH was defined by the experimental design, showing that inulinase immobilization is a promising biotechnological application of carbon nanotubes.